The major aim of this project is to elucidate mechanisms controlling cell fate decisions in developing T cells. Precursor T cells undergo a testing process in the thymus to ensure that cells expressing useless or self-reactive T cell antigen receptors (TCR) do not mature. These selection processes (negative and positive selection) require TCR engagement with peptide-MHC ligands expressed by thymic stromal cells. Quantitative aspects of TCR signaling determine whether the thymocyte will survive and mature or die. Thymocytes also receive signals as they mature that direct them to specific lineages. Expression and signaling through the gamma-delta TCR or the pre-TCR impose a bias on lineage choice that determines whether early T cell precursors specify the gamma-delta or alpha-beta T cell fate. Immature thymocytes that choose the alpha-beta pathway must then express a mature alpha-beta TCR that promotes positive selection and continued maturation but avoids negative selection. Our studies and those of others indicate that the strength and/ or duration of TCR signaling determine whether thymocytes will mature in the CD4 or CD8 T cell lineage. In addition, T cell fate may be determined by the integration of TCR signals with other developmental cues, such as those regulated by the transmembrane receptor, Notch.[unreadable] [unreadable] In efforts to understand the role of Notch signaling in T cell development, we have generated conditional null mutations of Presenilin1/2, targeting gene deletion to immature T cells. In this model, the generation of mature CD4 T cells is diminished in mice expressing a normal diverse TCR repertoire, but in mice expressing a class II-specific TCR transgene, production of CD4 T cells is almost completely blocked. Precursor thymocytes of the mutant express normal levels of TCR and the activation marker, CD69, but the level of CD5 (a negative regulator of TCR signaling) is dramatically reduced. These findings suggested that diminished TCR signaling during positive selection is responsible for the decreased generation of CD4 T cells in Presenilin mutant mice. Indeed, Presenilin-deficient thymocytes fluxed calcium poorly in response to TCR stimulation in vitro, an indication that TCR signal transduction was defective. Moreover, positive selection of Presenilin-deficient thymocytes was improved by introduction of a higher affinity MHC ligand, demonstrating that diminished TCR signaling in the absence of Presenilin can be compensated for by increased TCR-MHC affinity. These findings suggest a model in which Presenilin-dependent Notch signaling influences positive selection and the development of alpha-beta T cells by modifying TCR signal transduction.[unreadable] [unreadable] Results indicating that TCR signaling was defective in PS-deficient mice led us to address whether TCR signaling is abnormal in thymocytes of transgenic mice expressing a constitutively active form of Notch (NotchIC) and if altered TCR signaling could explain the observed alterations in CD4/CD8 development (fewer CD4 T and more CD8 T cells). A thorough analysis of the phenotype of immature CD4+CD8+ thymocytes of NotchIC mice, in the absence of the selecting MHC, showed that the same markers that are normally upregulated by positive selection are also upregulated by activated Notch. These results implied some relationship between Notch activity and TCR signaling in thymocytes. Indeed, immature CD4+CD8+ thymocytes of NotchIC mice responded to TCR cross-linking better than controls, as measured by the ability to flux calcium and to up-regulate expression of activation markers in response to in vitro TCR stimulation.[unreadable] [unreadable] Previous studies of mice with a diverse TCR repertoire indicated that NotchIC directs CD4+CD8+ thymocytes to adopt the CD8 lineage fate, at the expense of the CD4 lineage fate. However, it had not been determined whether NotchIC can direct thymocytes bearing a single MHC class II-restricted TCR to adopt the CD8 lineage. To assess the role of Notch in CD4/8 lineage commitment, NotchIC mice were crossed with mice expressing MHC class II-restricted TCR alpha-beta transgenes. Thymuses of control AND TCR or 5CC7 TCR transgenic mice generated CD4 T cells, and few if any CD8 T cells, as expected for MHC class II-selected thymocytes. In striking contrast, the thymus of NotchIC/ AND TCR or 5CC7 TCR mice produced no mature T cells. This result was unexpected since CD4+CD8+ thymocytes of NotchIC mice respond better than wild type mice to TCR stimulation in vitro. We considered the possibility that TCR signaling in vivo was so enhanced by activated Notch that it led to negative selection. However, attempts to reduce the quantity of selecting MHC had no discernable effect on the generation of mature T cells.[unreadable] [unreadable] The issue raised by these results is how altered TCR signaling in CD4+CD8+ thymocytes of Notch mutant mice is related to the alterations in CD4 and CD8 T cell development. If results from the Presenilin-deficient and NotchIC transgenic mice are compared, the effects of gain- and loss-of-Notch function on TCR signaling at the CD4+CD8+ stage are reciprocal, since inhibiting Notch activation diminishes TCR signaling, while enhancing Notch activity appears to potentiate it. However, both class II-restricted TCR/ Presenilin-deficient or /NotchIC transgenic mice show severely diminished production of CD4 T cells. Thus, the effect of constitutive Notch activity on the development of mature T cells cannot be explained by arguing that Notch promotes stronger TCR signaling, and stronger TCR signaling in turn favors the CD4 fate since no T cells are generated in class II-restricted TCR/ Notch IC transgenic mice. Although we do not as yet have a complete understanding of these results, the collective findings caused us to reconsider models for how Notch functions in the CD4/CD8 lineage decision. Our data indicate that the effect of inhibiting endogenous Notch on the generation of mature T cells is a consequence of modulating TCR signaling in CD4+CD8+ thymocytes rather than a direct effect on lineage commitment. These results reveal the danger in analyzing only gain-of-function mutations. Some but not all findings from NotchIC transgenic mice may represent normal physiology, perhaps because Notch is constitutively active and may not always co-regulate gene expression appropriately or with the right timing.